Objective To establish an effective PCR assay for leptospirosis detection , and applicate the assay in tree shrew, mongolian gerbil and gray hamster .Methods Sequence of leptospira was obtained from the NCBI Genbank , and primers were designed based on the sequences .The positive amplified fragments were sequenced to verify the reliability of the method.The samples from tree shrew, mongolian gerbils and hamsters were tested using this PCR method .Results The PCR method for detection of leptospirosis was successfully established .The positive rate of Leptospira was 8.33% in 60 samples of conventional tree shrews , 100% in 104 samples of the conventional Mongolian gerbils , and 0% in 60 samples of clean gray hamsters.Conclusions The establishment of this PCR assay is useful in the detection of leptospirosis in tree shrew, mongolian gerbil and gray hamster .The results of our investigation of leptospira infection levels of the three new experimental animals may promote their application in biomedical research .

Carcinoma, Non-Small-Cell Lung ; pathology ; surgery ; Humans ; Lung Neoplasms ; pathology ; surgery ; Neoplasm Staging ; Radiation Dosage ; Radiosurgery ; Treatment Outcome

Objective To develop a TaqMan MGB probe-based,sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Helicobacter hepaticus.Methods Primers and probes specific toflaB gene of Helicobacter hepaticus were designed.A TaqMan MGB probe-based,real-time fluorescence quantitative PCR was established.The specificity,sensitivity and stability of the assay were assassed.Then,the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Helicobacter hepaticus in 1081 clinical specimens during 2008-2011,compared with bacterial isolation and culture method and conventional PCR assay.Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high and there were no cross-reactivity with Helicobacter pylori,Campylobacter jejuni,Clostridium piliforme,Pasteurella pneumotropica,Escherichia coli,Pseudomonas aeruginosa.The detection limits was 8.3 copies.The correlation coefficient and slope value of standard curve were 0.999 and -3.227,respectively and the efficiency of TaqMan MGB-based probe realtime fluorescence quantitative PCR assay was 100％.The TaqMan MGB-based probe real-time fluorescence quantitative PCR and conventional PCR were preformed to detect Helicobacter hepaticus in 1081 clinical specimens,a total of 86 specimens were positive for Helicobacter hepaticus.However,there was only 4 specimens were positive by bacteria isolation and culture method.The results showed that TaqMan MGB -based probe real-time fluorescence quantitative PCR for Helicobacter hepaticas was more sensitive than bacteria isolation and culture method,and it could detect Helicobacter hepaticus DNA from clinical specimens directly,and detection time is only 2 hours.Conclusion The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was a reliable,specific,sensitive and useful tool for rapid detection of Helicobacter hepaticus.

Objective To improve the accuracy of detection through analyzing the phenotypes of P.pneumotropica isolates in laboratory animals in Beijing area .Methods 306 suspicious P.pneumotropica strains were identified by biochemical identification and 16S rDNA sequencing.Then, to obtain the phylogenetic relationships combined with colony characteristics on blood agar plates and biochemical characteristics of 53 biotypes .Results BD Phoenix 100 automated bacterial identification system and 16S rDNA sequencing identified P.pneumotropica positive rate of 306 isolates were 164/306 and 227/306, respectively.There were 140 phenotypes in 227 true-positive strains, of which 106 were biotype Heyl and 23 were biotype Jawetz .Conclusions In the samples of laboratory animals in Beijing area , P.pneumotropica infection mainly are of biotype Heyl , and less is of biotype Jawetz .The phenotypes are diverse and widely distributed .

Objective To develop a multiplex polymerase chain reaction ( mPCR) assay for detection of four pathogenic dermatophytes [Trichophyton mentagrophytes (Tm), Microsporum gypseum (Mg), Microsporum canis (Mc), and Arthroderma simii ( As) ] in laboratory animals, which could be used rapidly and simultaneously for direct detection of those four pathogens.Methods We designed 5 specific primers according to 18S-28S rRNA sequences of the four pathogenic dermatophytes reported in Genbank. The four mPCR assays were established through optimizing the concentration of primers, dNTP, TaqDNA polymerase and the annealing temperature.After verifying the specificity and sensibility, this method was used to detect 15 hair samples with artificial infection and 260 samples taken from laboratory animals.Results This mPCR technique can distinguish the four dermatophytes by producing 192 bp( Tm) ,460 bp( Mg) , 290 bp( Mc) and 602 bp( As) fragments.The sensibility for detection of the four dermatophytes was 5.9 pg/μL, 6.6 pg/μL, 9.5 pg/μL and 5.1 pg/μL, respectively.The results of 15 artificial infection samples were accurate, and the results of 260 hairs samples were negative for the four fungi.Conclusions Our results suggest that the mPCR assay developed in this study can efficiently detect the four dermatophytes, is a useful and rapid technique for rapid detection of the pathogenic dermatophytes in laboratory animals.

Objective To establish a rapid, simple, sensitive, and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews.Methods Specific primers and probes were designed, according to the HipO gene of Campylobacter jejuni, inV gene of Salmonella and ipaH gene of Shigella.The primers were confirmed by single pathogen quantitative PCR, and the sensitivity and specificity of the multiplex PCR were analyzed.Finall, the samples of experimental tree shrews were detected by this multiplex PCR method.Results The PCR element of TaqMan-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni, Salmonella or Shigella.Appropriate standard amplification curves of Campylobacter jejuni, Salmonella and Shigella in the multiplex quantitative PCR were obtained.The sensitivity of this method was 1×103 ng/μL.There was no false positive detection from other bacterial strains.Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.

Objective To evaluate the endovascular treatment of diffuse aortoiliac occlusive diseases. Methods Thirty-two patients underwent endovascular treatment in which rest pain was found in 84.38%, foot local gangrene in 15.62%. Mean age was 69.7 years (range, 52 years to 81 years) and 71.9% was male. Trans Atlantic Inter-Society Consensus C and D disease was respectively in 40.6% and 59.4% patients. Mean length of vasculopathy was (14.6 ± 1.2) cm (range, 4.5 cm to 19.5 cm) All patients had prohibitive risk for open revascularization. With the approach from femoral artery or brachial artery, combined techniques, such as recanahzation, balloon dilation, stent placement and concomitant common femoral endarterectomy were used. Results Technical success was achieved in twenty-nine patients(90.63%). The complication rate was 3.45%. Clinical status was markedly improved in eight cases (27.59%) and moderately improved in twenty-one cases(72.41%). Mean postoperative ABI was 0.73 ± 0.12, mean preoperative ABI was 0.32 ± 0.09. Significant differences were seen between postoperative ABI and preoperative ABI(P<0.05). Mean time of follow-up was (13.9±6.2) months. At 6 months, primary patency was 81.82% and secondary patency was 89.09%. At 12 months, primary patency was 63.64% and secondary patency was 80.18%. Conclusion Combined multiple endovascular technique is a safe and effective method in the treatment of poor risk diffuse aortoiliac occlusive diseases.

Objective To establish an effective PCR assay for detection of Bartonella, and application of this assay in tree shrew .Methods Sequence of Bartonella was obtained from NCBI Genbank .Three pairs of primers were designed based on this sequence .One pair of primers was determined through amplifying the major strains in China .Sixty tree shrew blood samples were tested with this PCR assay .The positive amplified fragments were sequenced to verify the reliability of this method .Results A PCR method for detection of Bartonella is successfully established , with a high specificity and the sensitivity was of 2.0 ×10 -5 μg/mL.Among the tested 60 blood samples , 15 positive cases were detected.Sequencing of the samples confirmed a 25%infection rate of Bartonella in the tree shrews, well consistent with the amplification results , and verified the applicability of this detection method .Conclusion The establishment of this method provides the basis for detection of Bartonella in tree shrew.

Objective To understand the Salmonella detectability in the laboratory animal testing laboratories, im-prove the level of detection for the quality of laboratory animals, by means of laboratory animals Salmonella proficiency tes-ting program.Method According to the proficiency testing program approved by CNAS, freeze-dried animal stool samples containing Salmonella bacteria and interference bacteria were prepared, and through stability and homogeneity tests quali-fied as proficiency testing samples.The randomly numbered samples were issued to the participating units by cold-chain transportation, and attached work instructions.The original reports and copies of the tests should be submitted on time.The sample results consistent with the results of the pretested results were considered as satisfactory, and the results inconsistent or fails to submit were judged as unsatisfactory.Results A total of 30 laboratories from 20 provinces and cities nationwide participated in this proficiency testing programs for Salmonella, including 28 ( 93.3%) laboratories with satisfactory re-sults, and two laboratories unsatisfactory ( 6.7%) .29 laboratories used separate culture methods, and two laboratories used PCR method.Conclusions The laboratory animal quality inspection agencies have good detection ability for Salmo-nella.The implementation of the capacity verification plan can well reflect the detection level of laboratories.

Objective To verify the detection ability of experimental animal quality detection laboratories in China for Staphylococcus aureus.Methods The testing samples for Staphylococcus aureus detection were prepared by bacterial culture, homogeneity test and stability test, according to the study plan approved by CNAS.Then the samples and operation instruction were sent to the participant laboratories.The detection reports from these laboratories should be submitted before the deadline expires, and the collected data were summarized and analyzed.Results There were 28 laboratories which joined to this test plan.Among them 22 laboratories ( 78.57%) achieved satisfactory test results, and six laboratories (21.43%) had unsatisfactory test results.27 Laboratories used the national standard detection assay, while only one labo-ratory used PCR assay.Conclusions Most of experimental animal quality testing laboratories in China have sufficient pro-ficiency in detection of Staphylococcus aureus.The obtained information are very helpful for the laboratory ability verification testing in future.